AG 50W and AG MP-50 strong acid cation
exchange resins are useful for single step purification
methods, for concentrating cationic solutes, and for
analytical determinations of various mixed cationic
solutes.
Section 2
Technical Description
Strong acid cation exchange resin is available as
Analytical Grade AG 50W resin, AG MP-50
macroporous resin, and Biotechnology Grade AG 50W
resin. The Analytical Grade AG 50W resin has been
exhaustively sized, purified, and converted to make it
suitable for accurate, reproducible analytical techniques.
Biotechnology Grade AG 50W resin is analytical grade
resin which is certified to contain less than 100
microorganisms per gram of resin.
AG 50W strong acid cation exchange resin is
composed of sulfonic acid functional groups attached to
1
a styrene divinylbenzene copolymer lattice. The amount
of resin crosslinking determines the bead pore size. A
resin with a lower crosslinkage has a more open structure
permeable to higher molecular weight substances than a
highly crosslinked resin. It also has a lower physical
resistance to shrinking and swelling, so that it absorbs
more water and swells to a larger wet diameter than a
highly crosslinked resin of equivalent dry diameter. For
example, typical applications of AG 50W-X2 2%
crosslinked resin and AG 50W-X4 4% crosslinked resin
include separation or concentration of peptides,
nucleotides, and amino acids. In high percentage
crosslinkage, (AG 50W-X8 8% resin, AG-50W-X12
12% resin, and AG 50W-X16 16% resin) applications
include separation of small peptides and amino acids,
removal of cations, and metal separations. Table 1 shows
the approximate molecular weight exclusion limits in
water for resins of various crosslinkages. All AG 50W
resins are supplied in the hydrogen form, and selected
AG 50W-X8 resins are available in sodium and
ammonium forms.
Table 1. Approximate Molecular Weight Exclusion
Limits for Ion Exchange Resins in Water
PercentApproximate MW Exclusion Limit
Crosslinkingfor Globular Molecules
2%2,700
4%1,400
8%1,000
10%800
12%400
AG MP-50 resin is the macroporous equivalent of
AG 50W resin. Its effective surface area approximates
35 square meters per dry gram, or 30-35% porosity.
The physical properties of the resins are listed in
Table 2. The cation exchange resins are thermally stable
and resistant to solvents (alcohols, hydrocarbons, etc.),
reducing agents, and oxidizing agents.
2
3
Table 2. Summary of the Properties of AG 50
and AG MP 50 Resins
In an ion exchange procedure, the counterions on the
resin are replaced by sample ions that have the same
charge. In applications involving a cation exchange
resin, such as AG 50 resin, neutral molecules and anions
do not interact with the resin. AG 50 resin is available
+
with H
, Na+, or NH
converted from one ionic form to another. Usually the
resin is used in an ionic form with a lower selectivity for
the functional group than the sample ions to be
exchanged. The sample ions are then exchanged when
introduced, and can be eluted by introducing an ion with
higher affinity for the resin or a high concentration of an
+
counterions. A resin can be
3
ion with equivalent or lower affinity. Table 3 shows the
relative selectivity of various counterions. In general, the
lower the selectivity of the counterion the more readily it
exchanges for another ion of like charge. The order of
selectivity can also be used to estimate the effectiveness
for different ions as eluants, with the most highly
selective being the most efficient. Finally, the order of
selectivity can be used to estimate the difficulty of
converting the resin from one form to another.
Conversion from a highly selected to a less highly
selected form requires an excess of the new ion.
4
5
Table 3. Relative Selectivity of Various
Counterions
Counterion for AG 50W-X8 Resin Counterion for AG 50W-X8 Resin
+
H
+
Li
+
Na
+
NH
4
+
K
+
Rb
+
Cs
+
Cu
+
Ag
2+
Mn
2+
Mg
Relative SelectivityRelative Selectivity
1.0Fe
0.85Zn
1.5Co
1.95Cu
2.5Cd
2.6Ni
2.7Ca
5.3Sr
7.6Hg
2.35Pb
2.5Ba
2+
2+
2+
2+
2+
2+
2+
2+
2+
2+
2+
2.55
2.7
2.8
2.9
2.95
3.0
3.9
4.95
7.2
7.5
8.7
Large mesh material (20-50 and 50-100 mesh) is
used primarily for large preparative applications and
batch operations where the resin and sample are slurried
together. Medium mesh resin (100-200 mesh) is used
primarily in column chromatography for analytical and
laboratory scale preparative applications. Fine mesh
material (200-400 and minus 400 mesh) is used for high
resolution analytical separations.
The AG 50 resins are available in several particle
size ranges. The flow rate in a chromatographic column
increases with increasing particle size. However, the
attainable resolution increases with decreasing particle
size and narrower size distribution ranges. Particle size is
given either in mesh size or micron size. The larger the
mesh size number, the smaller the particle size. Table 4
shows wet mesh and equivalent micron diameters.
6
Section 4
Resin Conversion
Table 5 outlines common techniques for converting
ion exchange resins from one ionic form to another.
Resin conversion is most efficiently carried out in the
column mode. However, when choosing a column,
7
remember that the resin may shrink, or it may swell as
much as 100%, depending on the conversion.
Conversions to ionic forms not listed in Table 5 can
be achieved using the information supplied in Table 3,
which lists relative selectivities of various counterions
for AG 50 resin. To convert a resin to an ionic form with
a higher selectivity, wash the resin with 2-3 bed volumes
of a 1 M solution of the desired counterion. For
conversion to an ionic form with a lower relative
selectivity for the resin, the necessary volume of
counterion solution will depend on the difference in
selectivity. As a general rule, use 1 bed volume of 1 M
counterion solution for each unit difference in relative
selectivity. For example, converting AG 50W-X8 resin
from the K
+
form (relative selectivity 2.5) to the H+form
(relative selectivity 1.0) would require 2-3 bed volumes
of 1 M HCl. The conversion is complete when all the K
ions are displaced by the H+ions.
8
Table 5. Techniques for AG Resin Conversion
Bio-Rex
®
AG 50 resin MSZ 50 resin
ConversionH
➝ Na
H+➝ pyridinium
+
+
from ➝ to
Reagent used1 M NaOH1 M pyridine
(wash with H
before pyridine)
O
2
Volumes of sol’n/22
vol. of resin
Flow rate
ml/min/cm
Type of exchange
Test for completeness pH 9
(2)
2
of bed
21
(1)
NN
(3)
—
of conversion
+
Rinse: vol. DI water/4—
vol. resin
Test for completion pH<9—
of rinsing
1. N = Neutralization
2. For 50-100 or finer mesh resin. For 20-50 mesh, about
rate is recommended.
3. Test for pH 4.8 – pH paper or methyl orange (red pH 1, yellow
pH 4.8). Test for pH 9 – pH paper or thymolphthalein (blue pH 10,
colorless at pH 9).
9
1
⁄5 the flow
Section 5
Instructions for Use
AG 50 and AG MP-50 resin may be used in either a
batch method or a column method. The batch method
consists of adding the resin directly to the sample and
stirring. The column method requires preparing a column
filled with resin, and passing the sample through.
5.1 Batch Method
The batch method is performed by adding the resin
directly into the sample and stirring. The resin should be
in the correct ionic form prior to beginning.
1. Weigh out about 5 grams of resin for every 100 ml of
sample. For larger scale applications or when an
exact amount of resin is needed, calculate the resin
volume based on the resin capacity.
2. Add resin to the sample and stir or shake gently for 1
hour.
3. Filter or decant the sample from the resin.
5.2 Column Method
The column method involves pouring a column with
the resin and passing the sample through to achieve the
separation. Particle size will determine the flow rate,
which will affect the separation. The resin should be in
the correct ionic form and equilibrated prior to adding
the sample.
1. Calculate the amount of resin required based on the
expected resin capacity and sample concentration. If
the sample ionic concentration is unknown, begin
with 5 grams of resin for 100 ml of sample, and then
optimize the volumes after obtaining the results.
2. Insure that the resin is in the ionic form which will
allow the sample ions to be exchanged onto the resin.
If conversion of the resin into another ionic form is
necessary, use the guidelines described above for
resin conversion (see Table 5).
3. Prepare the initial buffer, so that the pH and ionic
concentration will allow the sample ions to be
exchanged onto the column. For unknown solutions,
use deionized water.
10
11
4. Slurry and pour the resin into the column. Equilibrate
the resin in the initial buffer using 3 bed volumes of
buffer. Poorly equilibrated resin will not give
reproducible results. Alternatively, equilibration can
be done by the batch technique, prior to pouring the
column. First, convert the resin to the appropriate
form, then suspend it in the starting buffer. Check the
pH with a pH meter while stirring continuously.
Adjust the pH by adding acid or base dropwise to the
buffer until the desired pH is obtained. Then transfer
the resin to the column, and pass 1 bed volume of the
starting buffer through the column.
5. Add the initial buffer and allow excess buffer to pass
through the column, leaving enough buffer to just
cover the top of the resin bed.
6. Apply the sample dropwise to the top of the column
without disturbing the resin bed. Drain the sample
into the top of the bed and apply several small
portions of starting eluant, being very careful to rinse
down the sides of the column and to avoid stirring up
the bed. Drain each portion to the level of the resin
bed before the next portion is added. Never allow the
liquid level to drain below the top of the resin bed.
7. The actual flow rate that is used will depend upon the
application, the resin, and the column cross section.
To obtain flow rates for any given size column,
multiply the suggested flow rates in Table 6 by the
column cross-sectional area. Table 6 gives typical
flow rates of analytical grade resins.
8. If a cation-free solution is the goal, collect the effluent.
If the concentrated cations are of interest, allow all of
the sample to pass through the column, then elute the
metals with a solution containing a counterion of
higher selectivity than the bound cation.
Table 6. Suggested Flow Rates for Ion
Exchange Resin Columns
Flow Rates
Applicationcm/min
Removing trace ions5-10
Separations with very few components1-3
Separations of multi-component samples0.3-1.0
Using high resolution resins
with small particle size0.1-0.2
12
13
Section 6
Sample Protocol for Cation
Exchange Resins
6.1 Determination of Total Salts in Tap
Water
Approximately 85% of the Continental United States
is afflicted with hard water (3 grains or greater/gal). The
following is a rapid method of determining the total ionic
content of tap water as well as a good illustration of the
potential of ion exchange techniques. If the water
containing dissolved ions is allowed to flow over a
cation exchange resin, the metal ions will be
quantitatively exchanged for the hydrogen ions of the
resin. These hydrogen ions will appear in the eluant and
may then be titrated with standardized NaOH. Because
of the electroneutrality of the dissolved salts, the
milliequivalents of cations also represent the
milliequivalents of salts.
6.2 Materials
AG 50W-X8 resin, 50-100 mesh, hydrogen form–10 grams
Econo-Column®chromatography column, 1.0 x 0.79 cm
Methyl orange indicator solution (0.1%)
20 mM NaOH standard solution
3 M HCl
Flask–250 ml
6.3 Protocol
1. Pass approximately 150 ml of tap water through the
resin column.
2. Discard the first 20 ml of effluent.
3. Collect a 100 ml aliquot of effluent in a 250 ml flask.
4. Titrate with 20 mM NaOH to methyl orange end
point (yellow).
5. Calculate the salt content from the equivalents of
base used.
6.4 Calculation
Meq dissolved salts = ml of base x normality of base.
14
15
6.5 Notes
The experimental error is only that inherent in the
titration procedure. The error due to the ion exchange
itself is less than that of the titration. To avoid the error
due to interfering carbonate ions, neutralize alkaline tap
water with 0.1 M HCl, one drop at at time, to the methyl
orange end point.
The column may be used several times before
regeneration is necessary. To regenerate the resin, wash
it by passing approximately 50 ml of 3 M HCl through
the column, followed by 75 ml of distilled water.
Section 7
Applications
Strong cation exchange resins are used for sample
preparation, metal separations, weak acid separations,
peptide separations, amino acid separations, and nucleotide
separations. Tables 7-10 summarize the applications.
16
Section 8
Storage
The resins are stable for at least 2 years when stored
in the original, unopened container at room temperature
and protected from ultraviolet light.
Section 9
Stability
The resins are stable in acid, base, and organic
solvents, and may be autoclaved. To prevent bacterial
growth during prolonged storage of a poured column,
use a preservative such as 0.05% sodium azide or
thimerosol or 20% organic solvent such as methanol or
ethanol.
Table 7. Cation Exchangers for Sample
Preparation
ApplicationResinReference
Cation removal from AG 50W-X8 Ochiai, M.,
monosaccharidesresin224 (1980).
Removal of cationsAG 50W-X8 Hoffer, E. M., Kothny, E. L. and
from sulfateresinAppel, B. R., Atmospheric
Environment, 13, 303 (1979).
17
J. Chromatog., 194,
Table 7 (Continued)
ApplicationResinReference
Metal removalAG 50W-X8 Siemer, D. D., Anal. Chem., 52,
Cyclic nucleotideAG 50W-X8 Schwartz, J. P., Morris, N. R. and
extractionresinBreckenridge, B. M., J. Biol.
Concentration ofAG 50W-X8 Tryfiates, G. P. and Sattsangi, S., J.
vitamin B-6resinChromatog., 227, 181 (1982).
Concentration ofAG 50W-X8 Ford, C. W., J. Sci. Food Agric., 35,
amino acidsresin881 (1984).
Removal of contami- AG 50W-X8 Auf’mkolk, M., Koehrle, J., Hesch,
nants from I
Concentration ofAG 50W-X8 Schwartz, D. P. and McDonough,
chloramphenicolresinF. E., J. Assoc. Off. Anal. Chem.,
Removal of ethidium AG 50W-X8 Rodriguez, R. L. and Tait, R. C.,
bromide from resinRecombinant DNA Techniques: An
plasmids
Concentration of AG 50W-X8 Linblad, W. J. and Diegelmann,
isomers of trans-2, resinR. F., J. Chromatog., 315, 447
3-cis-3,4-dihydroxyl-(1984).
L-proline
Isolation of neutral AG 50W-X8 Terry, R. C. and Simon, M., J.
and cationic resin; AGChromatog., 232, 261(1982).
metabolites1-X8 resin
125
resin1874 (1980).
Chem., 248, 2699 (1973); Kuo, W.,
Hodgins, D. S. and Kuo, J. F., J.Biol. Chem., 248, 2705 (1973).
resinR. D. and Cody, V., J. Biol. Chem.,
261, 11623 (1986).
67, 583 (1984).
Introduction, p. 153-154 AddisonWesley Publishing Company (1983).
18
Table 7 (Continued)
ApplicationResinReference
Deionization of AG 50W-X8 Wigfield, Y. Y. and Lanouette, M.,
N-nitro-sodiethanol- resinJ. Assoc. Off. Anal. Chem., 68,
amine1142 (1985).
Deionization ofAG 50W-X8 Cullen, M. P., Turner, C. and Haycarbohydratesresin; AGcock, G. B., J. Chromatog., 337, 29
Concentration ofAG 50W-X2 Kapian, B. B., Schachter, B. S.,
nucleotide fragments resinOsterburg, H. H., de Velis, J. S. and
Concentration of 3-AG 50W-X4 Robert, J. C. and Serog, P., Clin.
methyl-L-histidineresinChim. Acta, 142, 161 (1984).
Separation of adeno- AG 50W-X4 Miura, G. A. and Chiang, D. K.,
syl-L-methionine resinAnal. Biochem., 147, 217 (1985).
from amino-cyclopropane carboxylic acid
N-acetyl-L-[
purificationresin J. Biol.Chem., 262, 6350 (1987).
Nitrite determination AG 50W-X12 Kordorouba, V. and Pelletier, M.,
in meatresinMitt. Geb. Lebensmitteiunters.
Glycopeptide andAG 50W-X2 Nishikawa, Y., et al., J. Biol Chem.,
oligosaccharideresin263, 8270 (1988).
purification
Aldehyde and ketone AG 50W-X2 Rendina, A. R. and Cleland, W. W.,
separationresinAnal. Biochem., 117, 213 (1981).
Diethyl acetalAG 50W-X8 Cho, Y. K., et al., Biochemistry, 27,
purificationresin3320 (1988).
35
2-X8 resin(1985).
Finch, C. E., Biochemistry, 17,
5516 (1978).
S] Met AG 50W Martin, D. J. and Rubenstein, P. A.,
Hyg., 79, 90 (1988).
19
Table 7 (Continued)
ApplicationResinReference
Ammonia determina- AG 50W-X8 Forman, D. T., Clinical Chem., 10,
tion in plasmaresin497 (1964).
Metal removalAG 50W-X8 Graf, E., J. Agric. Food Chem., 31,
Boron cleanupAG 50W-X8 Gregorie, D., Anal. Chem., 59,